4. The haemoglobin molecule and thalassaemia Flashcards
At which stages does Hb synthesis occur?
• Begins in pro-erythroblast:
- 65% erythroblast stage
- 35% reticulocyte stage
Where is haem synthesised?
- In the mitochondria (which has the enzyme ALAS)
* Dependent upon Fe incorporation into the cell
Where is globin synthesised?
Cytoplasmic ribosomes
Which globin chains is HbA formed of?
- 2α and 2β globin chains
* Each chain has a haem molecule at its centre
What is ferroprotoporphyrin?
Combination of protoporphyrin ring with central iron atom
How is globin synthesised?
• There are various different types of globin
• Combine with haem to form different haemoglobin molecules
• There are 8 functional globin chains arranged in 2 clusters:
- β-cluster (b, g, d and e globin genes) on the short arm of chromosome 11
- α-cluster (a and z globin genes) on the short arm of chromosome 16
• During early embryogenesis, production of cells in mainly in the yolk sac
• Later, the main sites of production are the liver and spleen
• Zeta and epsilon chains are produced until 6-8 weeks, after which there is a switch to alpha globin chains (problem - manifests early on, alpha thalassaemia major)
• Switch of production to bone marrow occurs shortly after birth
What are the 3 haemoglobins present in a normal adult?
- HbA
- HbA2
- HbF
Which haemoglobin is present in sufficient quantity to be visible on a HPLC chromatogram?
- Glycated HbA
* Only up to approx. 5%
Describe the primary, secondary and tertiary structure of globin
- Primary - α-globin chains: 141aa, non-α-globin chains: 146aa
- Secondary - 75% α and β form a helical arrangement
- Tertiary - approximate sphere, hydrophilic surface (charged polar side chains), hydrophobic core, haem pocket
What does cooperativity refer to when talking about the O2 carrying capacity of Hb?
Binding of one molecule facilitates the second molecule binding
What is the P 50?
26.6mmHg
partial pressure of O2 at which Hb is half saturated with O2
What does the normal position of the oxygen-haemoglobin dissociation curve depend on?
- Concentration of 2,3-DPG
- H+ ion concentration (pH)
- Concentration of CO2 in red blood cells
- Structure of Hb
What can cause the oxygen-dissociation curve to shift right?
Easy oxygen delivery (Hb only saturated at higher PO2, so gives up oxygen more easily) • High 2,3-DPG • High H+ • High CO2 • HbS
What can cause the oxygen-dissociation curve to shift left?
Gives up oxygen less readily (Hb saturated at lower PO2, increased affinity)
• Low 2,3-DPG
• HbF
What are haemoglobinopathies?
- Genetic disorders characterised by a defect of globin chain synthesis
- Most common inherited single gene disorder worldwide
How can we classify thalassaemia?
- Look at the globin chain type affected
* Look at clinical severity (minor “trait”, intermedia, major)
How many genes have been identified on chromosome 16?
- 7 genes identified (alpha like genes)
- Only 3 expressed - ζ, α1, and α
- 3 pseudo genes (ω) and a 4th gene that does not produce a detectable protein
Which genes have been identified on chromosome 11?
- Beta like genes
- ε and Gγ are expressed in the embryo
- β and δ expressed during later development
- The expression of δ globin chains never reaches that of the other globin chains
What is beta thalassaemia?
- Deletion of mutation in β globin gene(s)
- Results in reduced or absent production of β globin chains
- Prevalence – mainly Mediterranean countries (Greece, Cyprus, Southern Italy), Middle-East, Africa, Southern China, South-East Asia
- Inherited in recessive Mendelian fashion
- Carriers are asymptomatic
What determines the severity of thalassaemia?
- Degree of suppression of globin chain synthesis
- Some mutations result in no globin production (β0)
- Others have decreased levels of production (β+)
- Inheritance of 2 β0 genes will give rise to a Major
- Inheritance of 2 β+ genes will give rise to an intermedia (a clinically milder form)
How can thalassaemia be diagnosed in the lab?
• Microcytic hypochromic blood picture in the absence of iron deficiency
• Relatively high RBC count compared to Hb
• Peripheral film shows target cells, poikilocytosis but no anisocytosis
• Beta: HbA2 is raised depending of severity (rarely >7%), and raised HbF
• No simple diagnostic process to diagnose alpha thalassaemia
- presumptive diagnosis can be made if microcytosis and hypochromia is seen in the absence of iron deficiency
• Globin chain synthesis/DNA studies - gold standard
What are Globin Chain synthesis/DNA studies?
- Gold standard
- Genetic analysis for β-thalassaemia mutations and Xmnl polymorphism (in β-thalassaemias) and α-thalassaemia genotype (in all cases)
- Can sequence the α globin gene
- β-thalassaemia can usually be seen by electrophoresis
- Blood film of beta thalassaemia trait will show microcytosis, hypochromia and occasional cells showing basophillic stippling
- Marked degree of hypochromia due to Hb reduction in thalassaemia patients
- Some degree of poikilocytosis, and we see target cell
What causes thalassaemia major and what does it lead to?
- Carry 2 abnormal copies of the beta globin gene (so no HbA can be made)
- Leads to severe anaemia
- Incompatible with life without regular blood transfusions
- Clinical presentation usually after 4-6 months of life
What does a peripheral blood film show?
- Extreme hypochromia, microcytosis and poikilocytosis
* Howell-Jolly and nucleated RBCs often present (due to splenectophy and hyperplastic bone marrow)
How many blood transfusions are needed for beta thalassaemia major?
- 2-3 units per month
* Regular transfusion support
What is the problem with regular blood transfusions and how is this overcome?
- Iron overloaded
* Iron chelators to remove excess iron
What are pappenheimer bodies and when are they seen?
- Iron deposits
- Result of long-term transfusion regimens
- Seen as coarse blue granules in the RBCs (Perls stain)
Apart from pappenheimer bodies, what other inclusion bodies can be seen in beta thalassaemia major?
Alpha globin precipitates
What are the clinical presentations of thalassaemia major?
• Severe anaemia presenting after 4 months (beta)
• Hepatosplenomegaly
• Bone marrow will show erythroid hyperplasia
• Many of the consequences due to extra-medullar haematopoiesis
also e.g. prominence of maxilla bones and separation of the teeth
What are the clinical features (and other possible complications) of beta thalassaemia?
- Chronic fatigue
- Failure to thrive
- Jaundice
- Delay in growth and puberty
- Skeletal deformity
- Splenomegaly
- Iron overload (due to ineffective erythropoiesis)
- Cholelithiasis and biliary sepsis
- Cardiac failure
- Endocripathies
- Liver failure
How can thalassaemia major be treated?
- Regular blood transfusions
- Iron chelations therapy
- Splenectomy
- Supportive medical care
- Hormone therapy
- Hydroxyurea to boost HbF
- Bone marrow transplant - curative
What infections are thalassaemia patients more susceptible to?
- Yersinia - siderophilic (Fe loving) bacterium
- Gram negative sepsis
(Splenectomised patients given prophylaxis)
When do you start iron chelation therapy?
- Years later or when serum ferritin >100mcg/l
* Audiology and opthalmology screening prior to starting
How much of a difference has iron chelation therapy made to survival rates?
- 1960s - survival of patients pass their 30s was <30%
* First drug in 70s/80s - survival of patients pass their 30s was 80%
What are the 3 different types of iron chelators and how are they taken?
- DFO - subcutaneous or IV infusion, 5 days a week, 8-12 hour infusion
- Deferiprone - orally, 3 times a day, effective in reducing myocardial iron
- Deferasirox - orally, once a day
How can you increase the effectiveness of iron chelation in patients with heavy Fe overload?
Combination therapy
What are the disadvantages of deferiprone?
- Take it very often
- Short plasma half life
- Unpredictable control of body iron
- Toxicity (e.g. zinc deficiency)
What are the disadvantages of deferasirox?
- Cardiac protection uncertain
* Toxicity (limited)
How can iron overload be monitored?
- Serum ferritin - >2500 associated with increased complications; check every 3 months if transfused, otherwise annually; acute phase protein, so can also increase in other situations
- Liver biopsy - rare
- T2 cardiac and hepatic (ferriscan) MRI to quantify [Fe] (e.g. <20ms relaxation of cardiac fibres suggestive of Fe deposition)
How does a ferriscan determine liver iron concentration?
- Normal liver [Fe] <3mg/g
- > 15mg/g associated with sever complications
- Check annually, or biannually if >20mg/g
What does coinheritance of the sickle beta globin molecule with beta thalassaemia lead to
- Sickling disorder, rather than a thalassaemia
- Blood film shows features of both
- HbS will be the dominant haemoglobin as little/no HbA
What is HbE beta thalassaemia?
- Common combination in South East Asia
- Clinically variable in expression - can be as sever as beta thalassaemia major
- Thalassaemia properties - reduction in globin chain production
What is alpha thalassaemia (and cause)?
- Deletion or mutation in alpha globin chain gene(s)
- Reduced or absent production of alpha globin chains
- Affects both feotus and adult as production starts early in embryogenesis
- Excess β and γ chains form tetramers of HbH and Hb Barts respectively
- Severity depends on number of globin genes affected - 4 in total
What are the traits of a thalassaemia carrier
- Carry a single abnormal copy of the beta globin gene (w.r.t. beta thalassaemia)
- Carry either one or two abnormal copies of the alpha globin gene (w.r.t. alpha thalassaemia
- Usually asymptomatic, usually diagnosed on the basis of mild anaemia
Describe the peripheral blood film of HbH disease
- Haemolytic element
- Microcytosis, anisoocytosis, poikilocytosis
- “Puddling” of haemoglobin within RBC
What does Hb electrophoresis show in HbH disease?
A fast band
What are the problems associated with treatment of thalassaemia in developing countries?
- Lack of awareness of the problems
- Lack of experience of health care providers
- Availability of safe, screened blood
- Cost and compliance with iron chelation therapy
- Availability and cost of bone marrow transplant
When should thalassaemia be screened for prevention/management?
- Extended family screening
- Pre-marital screening (compulsory in Cyprus)
- Discourage marriage between realtives
- Antenatal testing
- Pre-natal diagnosis
